Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems for several reasons. First, some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Second, recombinant expression cassettes are genomically integrated and therefore mitotically stable. Third, these yeasts are capable of secreting large amounts of recombinant proteins. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; Cregg et al., Bio/Technology 11:905, 1993; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555; and Raymond, U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
Previously described expression systems for methylotrophic yeasts rely largely on the use of methanol-inducible transcription promoters. The use of methanol-induced promoters is, however, problematic as production is scaled up to commercial levels. The overall volume of methanol used during the fermentation process can be as much as 40% of the final fermentation value, and at 1000-liter fermentation scale and above the volumes of methanol required for induction necessitate complex and potentially expensive considerations.
There remains a need in the art for additional materials and methods to enable the use of methylotrophic yeasts for production of polypeptides of economic importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such materials and methods as well as other, related advantages.
It is an object of the present invention to provide transcription promoter and terminator sequences for use in Pichia methanolica. It is a further object of the invention to provide materials and methods for obtaining constitutive expression of heterologous DNA in P. methanolica. It is also an object of the invention to provide methods for production of polypeptides in P. methanolica, which methods can be readily scaled up to industrial levels, and to provide materials that can be used within these methods. It is another object of the invention to provide materials and methods for obtaining constitutive transcription of heterologous DNA to produce recombinant proteins in P. methanolica. 
Within one aspect, the present invention provides an isolated DNA molecule of up to 5000 nucleotides in length comprising nucleotide 93 to nucleotide 1080 of SEQ ID NO:1.
Within a second aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising at least a portion of the sequence of SEQ ID NO:1 from nucleotide 93 to nucleotide 1092, wherein the portion is a functional transcription promoter; a second DNA segment encoding a protein of interest other than a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase; and a third DNA segment comprising a transcription terminator. Within one embodiment, the first DNA segment is from 900 to 1500 nucleotides in length. Within another embodiment, the first DNA segment is from 900 to 1000 nucleotides in length. Within an additional embodiment, the first DNA segment is substantially free of Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase gene coding sequence. The DNA construct may further comprise a selectable marker, preferably a Pichia methanolica gene, more preferably a Pichia methanolica ADE2 gene. The DNA construct may be a closed, circular molecule or a linear molecule. Within other embodiments, the DNA construct further comprises a secretory signal sequence, such as the S. cerevisiae alpha-factor pre-pro sequence, operably linked to the first and second DNA segments. Within additional embodiments, the third DNA segment comprises a transcription terminator of a Pichia methanolica AUG1 or GAP2 gene.
Within a third aspect of the invention there is provided a Pichia methanolica cell containing a DNA construct as disclosed above. Within one embodiment, the DNA construct is genomically integrated. Within a related embodiment, the DNA construct is genomically integrated in multiple copies. Within a further embodiment, the P. methanolica cell is functionally deficient in vacuolar proteases proteinase A and proteinase B.
Within a fourth aspect of the invention there is provided a method of producing a protein of interest comprising the steps of (a) culturing a P. methanolica cell as disclosed above whereby the second DNA segment is expressed and the protein of interest is produced, and (b) recovering the protein of interest from the cultured cell.
Within a fifth aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising a Pichia methanolica gene transcription promoter; a second DNA segment encoding a protein of interest other than a Pichia methanolica protein; and a third DNA segment comprising nucleotides 2095 to 2145 of SEQ ID NO:1.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.
The term xe2x80x9callelic variantxe2x80x9d is used herein to denote an alternative form of a gene. Allelic variation is known to exist in populations and arises through mutation.
A xe2x80x9cDNA constructxe2x80x9d is a DNA molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of DNA combined and juxtaposed in an arrangement not existing in nature.
A xe2x80x9cDNA segmentxe2x80x9d is a portion of a larger DNA molecule having specified attributes. For example, a DNA segment encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, that, when read from the 5xe2x80x2 to the 3xe2x80x2 direction, encodes the sequence of amino acids of the specified polypeptide.
The term xe2x80x9cfunctionally deficientxe2x80x9d denotes the expression in a cell of less than 10% of an activity as compared to the level of that activity in a wild-type counterpart. It is preferred that the expression level be less than 1% of the activity in the wild-type counterpart, more preferably less than 0.01% as determined by appropriate assays. It is most preferred that the activity be essentially undetectable (i.e., not significantly above background). Functional deficiencies in genes can be generated by mutations in either coding or non-coding regions.
The term xe2x80x9cgenexe2x80x9d is used herein to denote a DNA segment encoding a polypeptide. Where the context allows, the term includes genomic DNA (with or without intervening sequences), cDNA, and synthetic DNA. Genes may include non-coding sequences, including promoter elements.
The term xe2x80x9cisolatedxe2x80x9d, when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
xe2x80x9cOperably linkedxe2x80x9d, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
A xe2x80x9cpolynucleotidexe2x80x9d is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated xe2x80x9cbpxe2x80x9d), nucleotides (xe2x80x9cntxe2x80x9d), or kilobases (xe2x80x9ckbxe2x80x9d). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When these terms are applied to double-stranded molecules they are used to denote overall length and will be understood to be equivalent to the term xe2x80x9cbase pairsxe2x80x9d. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
A xe2x80x9cpolypeptidexe2x80x9d is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as xe2x80x9cpeptidesxe2x80x9d.
The term xe2x80x9cpromoterxe2x80x9d is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5xe2x80x2 non-coding regions of genes. Sequences within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, and transcription factor binding sites. See, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed., The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif., 1987.
A xe2x80x9cpro sequencexe2x80x9d is a DNA sequence that commonly occurs immediately 5xe2x80x2 to the mature coding sequence of a gene encoding a secretory protein. The pro sequence encodes a pro peptide that serves as a cis-acting chaperone as the protein moves through the secretory pathway.
A xe2x80x9cproteinxe2x80x9d is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are commonly defined in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The term xe2x80x9csecretory signal sequencexe2x80x9d denotes a DNA sequence that encodes a polypeptide (a xe2x80x9csecretory peptidexe2x80x9d) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A secretory peptide and a pro peptide may be collectively referred to as a pre-pro peptide.
All references cited herein are incorporated by reference in their entirety.
The present invention provides isolated DNA molecules comprising a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter. The invention also provides isolated DNA molecules comprising a P. methanolica GAPDH gene terminator. The promoter and terminator can be used within methods of producing proteins of interest, including proteins of pharmaceutical or industrial value.
The sequence of a DNA molecule comprising a Pichia methanolica glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter, coding region, and terminator is shown in SEQ ID NO:1. The gene has been designated GAP2. Those skilled in the art will recognize that SEQ ID NO:1 represents a single allele of the P. methanolica GAP2 gene and that other functional alleles (allelic variants) are likely to exist, and that allelic variation may include nucleotide changes in the promoter region, coding region, or terminator region.
The partial sequence of a second P. methanolica glyceraldehyde-3-phosphate dehydrogenase gene, designated GAP1, is shown in SEQ ID NO:2.
Within SEQ ID NO:1, the GAPDH open reading frame begins with the methionine codon (ATG) at nucleotides 1093-1095. The transcription promoter is located upstream of the ATG. Gene expression experiments showed that a functional promoter was contained within the ca. 1000 nucleotide 5xe2x80x2-flanking region of the GAP2 gene.
Preferred portions of the sequence shown in SEQ ID NO:1 for use within the present invention as transcription promoters include segments comprising at least 900 contiguous nucleotides of the 5xe2x80x2 non-coding region of SEQ ID NO:1, and preferably comprising nucleotide 93 to nucleotide 1080 of the sequence shown in SEQ ID NO:1. Those skilled in the art will recognize that longer portions of the 5xe2x80x2 non-coding region of the P. methanolica GAP2 gene can also be used. Promoter sequences of the present invention can thus include the sequence of SEQ ID NO:1 through nucleotide 1092 in the 3xe2x80x2 direction and can extend to or beyond nucleotide 1 in the 5xe2x80x2 direction. In general, the promoter used within an expression DNA construct will not exceed 1.5 kb in length, and will preferably not exceed 1.0 kb in length. In addition to these promoter fragments, the invention also provides isolated DNA molecules of up to about 3300 bp, as well as isolated DNA molecules of up to 5000 bp, wherein said molecules comprise the P. methanolica GAP2 promoter sequence.
As disclosed in more detail in the examples that follow, the sequence of SEQ ID NO:1 from nucleotide 93 to nucleotide 1080 provides a functional transcription promoter. However, additional nucleotides can be removed from either or both ends of this sequence and the resulting sequence tested for promoter function by joining it to a sequence encoding a protein, preferably a protein for which a convenient assay is readily available.
Within the present invention it is preferred that the GAP2 promoter be substantially free of GAP2 gene coding sequence, which begins with nucleotide 1093 in SEQ ID NO:1. As used herein, xe2x80x9csubstantially freexe2x80x9d of GAP2 gene coding sequence means that the promoter DNA includes not more than 15 nucleotides of the GAP2 coding sequence, preferably not more than 10 nucleotides, and more preferably not more than 3 nucleotides. Within a preferred embodiment of the invention, the GAP2 promoter is provided free of coding sequence of the P. methanolica GAP2 gene. However, those skilled in the art will recognize that a GAP2 gene fragment that includes the initiation ATG (nucleotides 1093 to 1095) of SEQ ID NO:1 can be operably linked to a heterologous coding sequence that lacks an ATG, with the GAP2 ATG providing for intuition of translation of the heterologous sequence. Those skilled in the art will further recognize that additional GAP2 coding sequences can also be included, whereby a fusion protein comprising GAP2 and heterologous amino acid sequences is produced. Such a fusion protein may comprise a cleavage site to facilitate separation of the GAP2 and heterologous sequences subsequent to translation.
In addition to the GAP2 promoter sequence, the present invention also provides transcription terminator sequences derived from the 3xe2x80x2 non-coding region of the P. methanolica GAP2 gene. A consensus transcription termination sequence (Chen and Moore, Mol. Cell. Biol. 12:3470-3481, 1992) is at nucleotides 2136 to 2145 of SEQ ID NO:1. Within the present invention, there are thus provided transcription terminator gene segments of at least about 50 bp, preferably at least 60 bp, more preferably at least 90 bp, still more preferably about 200 bp in length. The terminator segments of the present invention may comprise 500-1000 nucleotides of the 3xe2x80x2 non-coding region of SEQ ID NO:1. These segments comprise the termination sequence disclosed above, and preferably have as their 5xe2x80x2 termini nucleotide 2095 of SEQ ID NO:1. Those skilled in the art will recognize, however, that the transcription terminator segment that is provided in an expression vector can include at its 5xe2x80x2 terminus the TAA translation termination codon at nucleotides 2092-2094 of SEQ ID NO:1 to permit the insertion of coding sequences that lack a termination codon.
Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are well known in the art and are disclosed by, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Murray, ed., Gene Transfer and Expression Protocols, Humana Press, Clifton, N.J., 1991; Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd edition, John Wiley and Sons, Inc., NY, 1995; Wu et al., Methods in Gene Biotechnology, CRC Press, New York, 1997. DNA vectors, including expression vectors, commonly contain a selectable marker and origin of replication that function in a bacterial host (e.g., E. coli) to permit the replication and amplification of the vector in a prokaryotic host. If desired, these prokaryotic elements can be removed from a vector before it is introduced into an alternative host. For example, such prokaryotic sequences can be removed by linearization of the vector prior to its introduction into a P. methanolica host cell.
Within certain embodiments of the invention, expression vectors are provided that comprise a first DNA segment comprising at least a portion of the sequence of SEQ ID NO:1 that is a functional transcription promoter operably linked to a second DNA segment encoding a protein of interest. When it is desired to secrete the protein of interest, the vector will further comprise a secretory signal sequence operably linked to the first and second DNA segments. The secretory signal sequence may be that of the protein of interest, or may be derived from another secreted protein, preferably a secreted yeast protein. A preferred such yeast secretory signal sequence is the S. cerevisiae alpha-factor (MFxcex11) pre-pro sequence (disclosed by Kurjan et al., U.S. Pat. No. 4,546,082 and Brake, U.S. Pat. No. 4,870,008).
Within other embodiments of the invention, expression vectors are provided that comprise a DNA segment comprising a portion of SEQ ID NO:1 that is a functional transcription terminator operably linked to an additional DNA segment encoding a protein of interest. Within one embodiment, the GAP2 promoter and terminator sequences of the present invention are used in combination, wherein both are operably linked to a DNA segment encoding a protein of interest within an expression vector.
Expression vectors of the present invention further comprise a selectable marker to permit identification and selection of P. methanolica cells containing the vector. Selectable markers provide for a growth advantage of cells containing them. The general principles of selection are well known in the art. The selectable marker is preferably a P. methanolica gene. Commonly used selectable markers are genes that encode enzymes required for the synthesis of amino acids or nucleotides. Cells having mutations in these genes cannot grow in media lacking the specific amino acid or nucleotide unless the mutation is complemented by the selectable marker. Use of such xe2x80x9cselectivexe2x80x9d culture media ensures the stable maintenance of the heterologous DNA within the host cell. A preferred selectable marker of this type for use in P. methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21). See, Raymond, U.S. Pat. No. 5,736,383. The ADE2 gene, when transformed into an ade2 host cell, allows the cell to grow in the absence of adenine. The coding strand of a representative P. methanolica ADE2 gene sequence is shown in SEQ ID NO:3. The sequence illustrated includes 1006 nucleotides of 5xe2x80x2 non-coding sequence and 442 nucleotides of 3xe2x80x2 non-coding sequence, with the initiation ATG codon at nucleotides 1007-1009. Within a preferred embodiment of the invention, a DNA segment comprising nucleotides 407-2851 is used as a selectable marker, although longer or shorter segments could be used as long as the coding portion is operably linked to promoter and terminator sequences. In the alternative, a dominant selectable marker, which provides a growth advantage to wild-type cells, may be used. Typical dominant selectable markers are genes that provide resistance to antibiotics, such as neomycin-type antibiotics (e.g., G418), hygromycin B, and bleomycin/phleomycin-type antibiotics (e.g., Zeocin(trademark); available from Invitrogen(trademark) Corporation, San Diego, Calif.). A preferred dominant selectable marker for use in P. methanolica is the Sh bla gene, which inhibits the activity of Zeocin(trademark).
The use of P. methanolica cells as a host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565; and U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,736,383. Expression vectors for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. To facilitate integration of the expression vector DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences (e.g., AUG1 3xe2x80x2 sequences). Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (xcfx84) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
Integrative transformants are preferred for use in protein production processes. Such cells can be propagated without continuous selective pressure because DNA is rarely lost from the genome. Integration of DNA into the host chromosome can be confirmed by Southern blot analysis. Briefly, transformed and untransformed host DNA is digested with restriction endonucleases, separated by electrophoresis, blotted to a support membrane, and probed with appropriate host DNA segments. Differences in the patterns of fragments seen in untransformed and transformed cells are indicative of integrative transformation. Restriction enzymes and probes can be selected to identify transforming DNA segments (e.g., promoter, terminator, heterologous DNA, and selectable marker sequences) from among the genomic fragments.
Differences in expression levels of heterologous proteins can result from such factors as the site of integration and copy number of the expression cassette among individual isolates. It is therefore advantageous to screen a number of isolates for expression level prior to selecting a production strain. Isolates exhibiting a high expression level will commonly contain multiple integrated copies of the desired expression cassette. A variety of suitable screening methods are available. For example, transformant colonies are grown on plates that are overlayed with membranes (e.g., nitrocellulose) that bind protein. Proteins are released from the cells by secretion or following lysis, and bind to the membrane. Bound protein can then be assayed using known methods, including immunoassays. More accurate analysis of expression levels can be obtained by culturing cells in liquid media and analyzing conditioned media or cell lysates, as appropriate. Methods for concentrating and purifying proteins from media and lysates will be determined in part by the protein of interest. Such methods are readily selected and practiced by the skilled practitioner.
For production of secreted proteins, host cells having functional deficiencies in the vacuolar proteases proteinase A, which is encoded by the PEP4 gene, and proteinase B, which is encoded by the PRB1 gene, are preferred in order to minimize spurious proteolysis. Vacuolar protease activity (and therefore vacuolar protease deficiency) is measured using any of several known assays. Preferred assays are those developed for Saccdaromyces cerevisiae and disclosed by Jones, Methods Enzymol. 194:428-453, 1991. A preferred such assay is the APNE overlay assay, which detects activity of carboxypeptidase Y (CpY). See, Wolf and Fink, J. Bact. 123:1150-1156, 1975. Because the zymogen (pro)CpY is activated by proteinase A and proteinase B, the APNE assay is indicative of vacuolar protease activity in general. The APNE overlay assay detects the carboxypeptidase Y-mediated release of xcex2-naphthol from N-acetyl-phenylalanine-p-naphthyl-ester (APNE), which results in the formation of an isoluble red dye by the reaction of the xcex2-naphthol with the diazonium salt Fast Garnet GBC. Cells growing on assay plates (YEPD plates are preferred) at room temperature are overlayed with 8 ml Rxc3x97M. Rxc3x97M is prepared by combining 0.175 g agar, 17.5 ml H2O, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the mixture to dissolve the agar, cooling to xcx9c55xc2x0 C., adding 2.5 ml freshly made APNE (2 mg/ml in dimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and, immediately before assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The overlay is allowed to solidify, and color development is observed. Wild-type colonies are red, whereas CPY deletion strains are white. Carboxypeptidase Y activity can also be detected by the well test, in which cells are distributed into wells of a microtiter test plate and incubated in the presence of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and dimethylformamide. The cells are permeabilized by the dimethylformamide, and CpY in the cells cleaves the amide bond in the BTPNA to give the yellow product p-nitroaniline. Assays for CpY will detect any mutation that reduces protease activity so long as that activity ultimately results in the reduction of CpY activity.
P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25xc2x0 C. to 35xc2x0 C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto(trademark) Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto(trademark) yeast extract (Difco Laboratories), 0.004% adenine, 0.006% L-leucine).
For large-scale culture, one to two colonies of a P. methanolica strain can be picked from a fresh agar plate (e.g, YEPD agar) and suspended in 250 ml of YEPD broth contained in a two-liter baffled shake flask. The culture is grown for 16 to 24 hours at 30xc2x0 C. and 250 rpm shaking speed. Approximately 50 to 80 milliliters of inoculum are used per liter starting fermentor volume (5-8% v/v inoculum).
A preferred fermentation medium is a soluble medium comprising glucose as a carbon source, inorganic ammonia, potassium, phosphate, iron, and citric acid. As used herein, a xe2x80x9csoluble mediumxe2x80x9d is a medium that does not contain visible precipitation. Preferably, the medium lacks phosphate glass (sodium hexametaphosphate). A preferred medium is prepared in deionized water and does not contain calcium sulfate. As a minimal medium, it is preferred that the medium lacks polypeptides or peptides, such as yeast extracts. However, acid hydrolyzed casein (e.g., casamino acids or amicase) can be added to the medium if desired. An illustrative fermentation medium is prepared by mixing the following compounds: (NH4)2SO4 (1.5 grams/liter), K2HPO4 (2.60 grams/liter), KH2PO4 (9.50 grams/liter), FeSO4.7H2O (0.40 grams/liter), and citric acid (1.00 gram/liter). After adding distilled, deionized water to one liter, the solution is sterilized by autoclaving, allowed to cool, and then supplemented with the following: 60% (w/v) glucose solution (47.5 milliliters/liter), 10xc3x97 trace metals solution (20.0 milliliters/liter), 1 M MgSO4 (20.0 milliliters/liter), and vitamin stock solution (2.00 milliliters/liter). The 10xc3x97 trace metals solution contains FeSO4.7H2O (100 mM), CuSO4.5H2O (2 mM), ZnSO4.7H2O (8 mM), MnSO4.H2O (8 mM), CoCl2.6H2O (2 mM), Na2MoO4.2H2O (1 mM), H3BO3 (8 mM), KI (0.5 mM) NiSO4.6H2O (1 mM), thiamine (0.50 grams/liter), and biotin (5.00 milligrams/liter). The vitamin stock solution contains inositol (47.00 grams/liter), pantothenic acid (23.00 grams/liter), pyrodoxine (1.20 grams/liter), thiamine (5.00 grams/liter), and biotin (0.10 gram/liter). Those of skill in the art can vary these particular ingredients and amounts. For example, ammonium sulfate can be substituted with ammonium chloride, or the amount of ammonium sulfate can be varied, for example, from about 11 to about 22 grams/liter.
After addition of trace metals and vitamins, the pH of the medium is typically adjusted to pH 4.5 by addition of 10% H3PO4. Generally, about 10 milliliters/liter are added, and no additional acid addition will be required. During fermentation, the pH is maintained between about 3.5 to about 5.5, or about 4.0 to about 5.0, depending on protein produced, by addition of 5 N NH4OH.
An illustrative fermentor is a BIOFLO 3000 fermentor system (New Brunswick Scientific Company, Inc.; Edison, N.J.). This fermentor system can handle either a six-liter or a fourteen-liter fermentor vessel. Fermentations performed with the six-liter vessel are prepared with three liters of medium, whereas fermentations performed with the fourteen-liter vessel are prepared with six liters of medium. The fermentor vessel operating temperature is typically set to 30xc2x0 C. for the course of the fermentation, although the temperature can range between 27-31xc2x0 C. depending on the protein expressed. The fermentation is initiated in a batch mode. The glucose initially present is often used by approximately 10 hours elapsed fermentation time (EFT), at which time a glucose feed can be initiated to increase the cell mass. An illustrative glucose feed contains 900 milliliters of 60% (w/v) glucose, 60 milliliters of 50% (w/v) (NH4)2SO4, 60 milliliters of 10xc3x97 trace metals solution, and 30 milliliters of 1 M MgSO4. Pichia methanolica fermentation is robust and requires high agitation, aeration, and oxygen sparging to maintain the percentage dissolved oxygen saturation above 30%. The percentage dissolved oxygen should not drop below 15% for optimal expression and growth. The biomass typically reaches about 30 to about 80 grams dry cell weight per liter at 48 hours EFT.
Proteins produced according to the present invention are recovered from the host cells using conventional methods. If the protein is produced intracellulary, the cells are harvested (e.g., by centrifugation) and lysed to release the cytoplasmic contents. Methods of lysis include enzymatic and mechanical disruption. The crude extract is then fractionated according to known methods, the specifics of which will be determined for the particular protein of interest. Secreted proteins are recovered from the conditioned culture medium using standard methods, also selected for the particular protein. See, in general, Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York, 1994.
The materials and methods of the present invention can be used to produce proteins of research, industrial, or pharmaceutical interest. Such proteins include enzymes, such as lipases, cellulases, and proteases; enzyme inhibitors, including protease inhibitors; growth factors such as platelet derived growth factor (PDGF), fibroblast growth factors (FGF), epidermal growth factor (EGF), vascular endothelial growth factors (VEGFs); glutamic acid decarboxylase (GAD); cytokines, such as erythropoietin, thrombopoietin, colony stimulating factors, interleukins, and interleukin antagonist; hormones, such as insulin, proinsulin, leptin, and glucagon; and receptors, including growth factor receptors, which can be expressed in truncated form (xe2x80x9csoluble receptorsxe2x80x9d) or as fusion proteins with, for example, immunoglobulin constant region sequences. DNAs encoding these and other proteins are known in the art. See, for example, U.S. Pat. Nos. 4,889,919; 5,219,759; 4,868,119; 4,968,607; 4,599,311; 4,784,950; 5,792,850; 5,827,734; 4,703,008; 4,431,740; and 4,762,791; and WIPO Publications WO 95/21920 and WO 96/22308.
It is particularly preferred to use the present invention to produce unglycosylated pharmaceutical proteins. Yeast cells, including P. methanolica cells, produce glycoproteins with carbohydrate chains that differ from their mammalian counterparts. Mammalian glycoproteins produced in yeast cells may therefore be regarded as xe2x80x9cforeignxe2x80x9d when introduced into a mammal, and may exhibit, for example, different pharmacokinetics than their naturally glycosylated counterparts.